A preS2 aa1–26-specific humoural response marks functional cure in chronic HBV infection

BACKGROUND: Functional cure (FC) of chronic hepatitis B (CHB) is closely associated with restoration of HBV-specific humoural immunity, yet the epitope-resolved features of humoural immunity recovery during interferon-induced FC remain unknown. METHODS: We profiled HBV-specific humoural responses in 65 patients with CHB drawn from three independent PEG-IFNα-treated cohorts, including nucleos(t)ide analogue-treated patients with viral suppression and inactive HBsAg carriers. Linear epitope mapping was performed using phage immunoprecipitation sequencing (PhIP-seq), with key findings validated by longitudinal ELISA and complemented by ex vivo phenotypic characterisation of epitope-specific B cells using fluorescent peptide tetramers. FINDINGS: PhIP-seq identified 297 reactive peptides, with a marked enrichment of antibody reactivity toward the HBsAg preS domain in patients who achieved FC. Longitudinal analysis demonstrated that preS2-directed antibodies, particularly those targeting the N-terminal amino acids 1-26 (preS2 aa1-26), increased progressively and closely parallelled HBsAb seroconversion, consistently distinguishing FC from non-FC group. In parallel, ex vivo B-cell profiling revealed that preS2 aa1-26-specific B cells exhibited a plasmablast-skewed and IgG-dominant profile, in contrast to the more IgM-biased and less differentiated phenotypes observed in preS1- and SHBs-specific B cells. Within the preS2-specific compartment, FC showed higher CXCR5 and CD69 expression than non-FC group, indicative of enhanced maturation and improved follicular homing potential. INTERPRETATION: PreS2 aa1-26 may serve as a key humoural HBV-specific epitope associated with functional cure, with potential implications for immunotherapeutic strategies in CHB. FUNDING: This work was supported by the National Science and Technology Major Project (2025ZD01905905), National Key Research and Development Program of China (2022YFA1303600 and 2023YFC2308100), and the National Natural Science Foundation of China (82130019 and 82272311).